Trafficking Of The Mu-Opioid Receptor Following Chronic Morphine Treatment

Alexandra Cooke, Stuart Mundell, Graeme Henderson, Eamonn Kelly. University of Bristol, Department of Physiology and Pharmacology,, Bristol, BS8 1TD, UK

Sustained agonist activation of the μ-opioid receptor (MOPr) rapidly initiates regulatory events including, desentization and internalization, that are thought to contribute to the development of tolerance at the single cell level (Williams et al., 2001). The current study examined the molecular mechanisms that mediate the trafficking of the MOPr and its relationship to cellular tolerance, following chronic (72 h) morphine treatment.

In HEK 293 stably expressing haemagglutin (HA)-tagged MOPr (HEK-MOPr) we used ELISA to investigate the internalization and recycling of the MOPr, as previously described (Johnson et al., 2006). The extent of DAMGO-induced (10 μM; 30 min) internalization was significantly reduced in cells that had been exposed to 1 μM morphine for 72 h in comparison to control cells (14.6 ± 1.7% internalization v 25.2 ± 2.5% internalization, respectively; n = 5; Student’s t test; P<0.05). This effect was due to chronic morphine because in cells that had been exposed to 1 μM morphine for 24 h a reduction in DAMGO-induced internalization was not observed (n = 4; Student’s t test; P = 0.149). Furthermore, in cells that had been exposed to morphine chronically (72 h), the MOPr did not appear to recycle normally, only 0.8 ± 10.4 % of the DAMGO-internalized receptor had recycled back after 30 min in the presence of naltrexone (1µM), whereas, in control cells, 49.4 ± 8.4 % of the receptor had recycled back to the plasma membrane at 30 min (n = 5; Student’s t test; P< 0.01).

Chronic morphine treatment did not alter the expression of arrestins, dynamin 2 or G-protein-coupled receptor kinase 2 (GRK2) in whole cell lysates prepared from HEK-MOPr cells (n = 3). Preliminary data obtained so far by immunoprecipitating the HA-tagged receptor from the HEK-MOPr cells suggests there is a substantial decrease (<60% loss of receptor) in the cellular expression of the MOPr following chronic morphine treatment, an effect that is inhibited by the co-incubation of cells with naltrexone (1 µM) for 72 h. However, when looking at receptor expression by ELISA, we find only a 28% decrease in surface receptor expression following chronic (72h) morphine treatment (n = 5; one-sample t-test; P<0.01)), suggesting that the cell attempts to maintain the level of MOPr at the cell surface. Investigations into the location of the MOPr within the cell by immunofluorescence microscopy showed that following chronic (72h) morphine treatment, MOPr clusters form in the perinuclear region of the cell in addition to the receptor being present at the cell surface. Experiments are underway to identify the intracellular compartment where MOPr accumulates following chronic morphine treatment.

In summary, we find that following chronic morphine exposure, the high efficacy agonist, DAMGO, induces significantly less internalization. Interestingly, the MOPr also appears to be unable to recycle following chronic morphine treatment. Work is currently underway to investigate the molecular mechanism underlying these changes in MOPr trafficking.

Williams JT et al., (2001) Physiol Rev. 81: 299-343.

Johnson EA et al., (2006) Mol. Pharmacol. 70:676-685.